U.S. patent number 4,210,638 [Application Number 05/887,541] was granted by the patent office on 1980-07-01 for antiviral composition and method of treating virus diseases.
This patent grant is currently assigned to PCR, Inc., University of Miami. Invention is credited to Sheldon Greer.
United States Patent |
4,210,638 |
Greer |
July 1, 1980 |
Antiviral composition and method of treating virus diseases
Abstract
Pharmaceutical compositions suitable for the treatment of Herpes
or Herpes-like viruses are disclosed, wherein the compositions
contain 5-trifluoromethyl-2'-deoxycytidine and a cytidine deaminase
inhibitor. Also disclosed are methods of treating patients
suffering from a disease caused by a Herpes or Herpes-like virus,
with the method comprising administering to the patient a
therapeutically effective amount of
5-trifluoromethyl-2'-deoxycytidine and a cytidine deaminase
inhibitor.
Inventors: |
Greer; Sheldon (Miami, FL) |
Assignee: |
PCR, Inc. (Gainesville, FL)
University of Miami (Miami, FL)
|
Family
ID: |
25391373 |
Appl.
No.: |
05/887,541 |
Filed: |
March 17, 1978 |
Current U.S.
Class: |
514/49; 514/50;
536/28.5; 536/28.52 |
Current CPC
Class: |
C07H
19/06 (20130101) |
Current International
Class: |
C07H
19/06 (20060101); C07H 19/00 (20060101); A61K
031/70 (); C07H 017/00 () |
Field of
Search: |
;536/23 ;424/180 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Brown; Johnnie R.
Assistant Examiner: Hazel; Blondel
Attorney, Agent or Firm: Murray and Whisenhunt
Claims
What is claimed is:
1. A pharmaceutical composition for the treatment of Herpes or
Herpes-like viruses, comprising an effective amount of
5-trifluoromethyl-2'-deoxycytidine and an effective amount of a
cytidine deaminase inhibitor which is tetrahydrouridine or
2'-deoxytetrahydrouridine.
2. Composition of claim 1, wherein said
5-trifluoromethyl-2'-deoxycytidine is present in said composition
in an amount of about 0.01 to 50% by weight.
3. Composition of claim 1, wherein the weight ratio of said
cytidine deaminase inhibitor to said
5-trifluoromethyl-2'-deoxycytidine is from about 500:1 to 1:1.
4. Composition of claim 3, in a form suitable for intravenous
administration.
5. Composition of claim 3, wherein said
5-trifluoromethyl-2'-deoxycytidine is present in an amount of 0.05
to about 5% by weight.
6. Composition of claim 3, in a form suitable for topical
administration.
7. Composition of claim 5, wherein the amount of said
5-trifluoromethyl-2'-deoxycytidine in said composition is about 5
to 50% by weight.
8. Composition of claim 3, in a form suitable for oral
administration.
9. Composition of claim 8, wherein said
5-trifluoromethyl-2'-deoxycytidine is present in an amount of about
0.05 to 10% by weight.
10. A method of treating a host suffering from a Herpes or a
Herpes-like virus infection, said method comprising administering
to said host a therapeutically effective amount of
5-trifluoromethyl-2'-deoxycytidine and a therapeutically effective
amount of a cytidine deaminase inhibitor which is tetrahydrouridine
or 2'-deoxytetrahydrouridine.
11. Method of claim 10, wherein said inhibitor is administered to
the host prior to the administration of said
5-trifluoromethyl-2'-deoxycytidine.
12. A method according to claim 1 wherein the disease is caused by
a Herpes Simplex Virus type 1 or 2 or varicella-zoster virus.
13. A method according to claim 10, wherein the
5-trifluoromethyl-2'-deoxycytidine is administered to the host in
an amount of about 0.01 to 0.25 mmoles/kg of body weight/day.
14. A method according to claim 13, wherein the
5-trifluoromethyl-2'-deoxycytidine is administered to the host in
an amount of about 10 mg/kg body weight/day.
15. Method of claim 14, wherein said cytidine deaminase inhibitor
is administered such that the weight ratio of said inhibitor to
said 5-trifluoromethyl-2'-deoxycytidine is about 500:1 to 1:1.
Description
BACKGROUND OF THE INVENTION
The present invention relates to pharmaceutical compositions
containing 5-trifluoromethyl-2'-deoxycytidine and a cytidine
deaminase inhibitor, and to a method for treating diseases caused
by Herpes or Herpes-like virus by administering
5-trifluoromethyl-2'-deoxycytidine and a cytidine deaminase
inhibitor.
Diseases caused by Herpes and Herpes-like viruses are particularly
widespread in man. Examples of Herpes viruses are Herpes simplex
virus (HSV) Types 1 (HSV-1) and 2 (HSV-2) and Herpes
varicella-zoster virus (VZV) that causes chicken pox in children
and shingles in adults. Other examples of Herpes-like viruses are
Epstein-Barr virus, Pseudorabies virus, Cytomegalo virus, Marek's
disease virus of chickens, equine abortion virus (EAV) and
Lucke-frog virus.
Herpes simplex viruses are strongly implicated in many pathological
systems and include ocular (Keratitis), cutaneous (including
genital and oral), and systemic disseminated infections. One
disease caused by the Herpes simplex virus Type 1 (HSV-1) is a
particularly virulent form of encephalitis which, if not treated
effectively, is usually fatal. Recurrent and persistent genital
infections occur with HSV-2 that are widespread in the population
and defy management so that these patients suffer great physical
discomfort and psychological distress. HSV-1 causes substantial
discomfort to a large segment of the population. There is at this
time no known way to manage recurrent infections or to combat this
virus in its latent stage.
Varicella-zoster is often the cause of morbidity in
immunosuppressed patients such as kidney transplant recipients.
Cytomegalo virus causes embryological abnormalities, perinatal
neurological disease and great problems in the neonate; like
zoster, it is a neurotropic virus.
An extremely active area of the current medical research is the
study of virus caused diseases, in particular those induced by
Herpes and Herpes-like viruses. An important part of this research
is the development of selective antiviral agents for the treatment
of these diseases. As will be discussed in more detail below, the
major problem with the antiviral agents presently available is
their tendency to undergo catabolism in the body and, more
importantly, their toxicity towards uninfected cells; that is,
their nonselectivity.
The search for effective antiviral agents which exhibit specific
antiviral activity against cells infected with Herpes and
Herpes-like viruses has met with varying degrees of success. In
1962, Kaufman (IDU Therapy of Herpes Simplex, Arch. Ophthalmol. 67,
583, 1962) investigated the antiviral activity of certain
5-halo-deoxyuridine compounds and found tht 5-iodo-2'-deoxyuridine
(IdU) exhibits antiviral activity against HSV infections of the
eye. Subsequently, Heidelberger discovered that, while
5-fluorodeoxyuridine exhibits very little antiviral activity,
5-trifluoromethyl-2'-deoxyuridine, or 5-trifluoro thymidine
(F.sub.3 dT), does exhibit antiviral activity against infections of
the eye. The compound F.sub.3 dT is described and claimed in U.S.
Pat. No. 3,201,387. ##STR1##
Although IdU is effective against Herpes Keratitis it is less
effective than F.sub.3 dT and is not as effective in systemic
infections or in the treatment of Herpes genitalis.
Despite exhibiting antiviral activity, these two compounds (IdU and
F.sub.3 dT) suffer from two major disadvantages. The first is that
the compounds undergo rapid catabolism in the body which results in
significant reduction of antiviral effectiveness of the compound.
The second disadvantage is that the compounds exhibit toxicity
towards uninfected cells which, in turn, results in the generation
of unpleasant and harmful side effects. IdU has been abandoned for
the treatment of Herpes encephalitis because of its toxicity and
its ineffectiveness, and F.sub.3 dT has not been considered for the
treatment of systemic infections. There are some approaches that
involve direct intracranial injection of this compound for the
treatment of encephalitis; however, the studies are still at the
stage of animal models. Furthermore, the approach to treatment
appears to be associated with potential hazards for use in
humans.
Studies on various 5-substituted analogs of deoxyuridine, including
5-methyl amino-2'-deoxyuridine, 5-thiocyanato-2'-deoxyuridine,
5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine,
5-phenyl-2'-deoxyuridine and 5-allyl-2'-deoxyuridine have been
reported which indicate that these compounds do exhibit antiviral
activity against Herpes simplex in cell culture; however, the
success of these compounds will likely remain limited to cell
culture studies, in spite of the fact that they are non-toxic in
culture, for they are substrates for the catabolic enzymes uridine
and thymidine phosphorylase.
Adenine arabinoside has been shown to decrease the incidence of
death due to human encephalitis. However, the number of individuals
with neurological sequelae was discouraging. That is, the drug
decreased the mortality but increased the morbidity. Furthermore,
ara-A or ara-AMP is neither effective against recurrent genital
Herpes nor does it decrease the incidence of latent virus
infection. Phosphonacetic acid is effective in animal systems;
however it must be administered in most cases very soon after
infection, and is usually ineffective if the onset of treatment is
delayed to coincide with realistic intervals for consideration for
use in humans.
Other drugs such as ara-T, 4-amino-5-iodo-deoxyuridine and
acycloguanine are in various stages of development and are far from
being ready for use in clinical studies. Furthermore, in view of
the capacity of viruses to mutate to resistance to a drug (as is
the case with phosphonacetic acid) it is likely that ultimately
viral chemotherapy will involve a combination of drugs that act via
different mechanisms.
More recently, attention has turned to the study of deoxycytidine
compounds as possible antiviral agents and, in particular, the
5-substituted analogs thereof. Greer et al. (Annals of the New York
Academy of Sciences, Volume 255, 359, 1975) have studied the
antiviral activity of 5-halo-2'-deoxycytidines, namely
5-bromo-2'-deoxycytidine (BrdC) and 5-iodo-2'-deoxycytidine (IdC).
The studies have shown that these 5-halo-2'-deoxycytidine compounds
possess a similar antiviral activity against HSV infected cells as
that possessed by the corresponding 5-halo-2'-deoxyuridine
compounds, but most importantly that the 5-halo-2'-deoxycytidine
compounds are substantially less toxic towards uninfected cells
than the deoxyuridine compounds. Kurimoto et al. Folia. Ophthalmol.
Japan, 20, 49 (1969) have shown that IdC is more effective in the
treatment of Herpes Keratitis in humans than IdU.
A drawback of the 5-halo-2'-deoxycytidine compounds is their
tendency to undergo deamination in the presence of deaminating
enzymes, such as cytidine deaminase. Such enzymes are usually
present in the blood and catalyze the deamination of the
5-halo-2'-deoxycytidine compound to the corresponding
5-halo-2'-deoxyuridine compound. As a result of this deamination,
uridine compounds are formed which do not display selectivity and
which exhibit toxicity towards uninfected cells and generate
unpleasant and harmful side effects. Furthermore, deoxyuridine
analogs are further degraded to metabolites that do not display
antiviral activity.
In order to overcome this problem of deamination, it has been found
necessary to employ a deamination inhibitor, and tetrahydrouridine
(H.sub.4 U) and 2'-deoxytetrahydrouridine (H.sub.4 dU) have been
found particularly suitable for this purpose. These two compounds
are described in U.S. Pat. No. 4,017,606 (Hanze et al.). The patent
describes the synthesis of H.sub.4 U and H.sub.4 dU starting from a
compound whose general formula covers the compound
5-trifluoromethyl-2'-deoxycytidine(F.sub.3 methyl dC) which forms
the subject of the present invention. However, there is no specific
disclosure of F.sub.3 methyl dC in the Hanze et al. patent and
there is no disclosure of any utility of F.sub.3 methyl dC as an
antiviral agent.
Studies have been recently reported of the antiviral activity of
5-methyl-2'-deoxycytidine and 5-ethyl-2'-deoxycytidine. Shugar (J.
Med. Chem., Vol. 17, No. 3, 296, 1974) discovered that
5-ethyl-2'-deoxycytidine possesses only a low antiviral activity
against HSV infected cells and no activity against vaccinia and
vesicular stomatitis. Very recent studies by Lin and Prusoff
(Abstracts of Papers, 174th ACS Meeting, American Chemical Society,
Aug. 28-Sept. 2, 1977) have shown that 5-methyl-2'-deoxycytidine is
less effective as an antiviral agent against HSV infected cells
than 5-methyl-2'-deoxyuridine.
SUMMARY OF THE INVENTION
The compound 5-trifluoromethyl-2'-doxycytidine (also called F.sub.3
methyl dC) having the formula: ##STR2## exhibits several surprising
and unexpected advantages over the prior antiviral agents discussed
above. In particular, the compound F.sub.3 methyl dC exhibits an
increased specificity towards cells infected with Herpes and
Herpes-like viruses. It is not anabolized to a cytotoxic metabolite
in uninfected cells. Furthermore, F.sub.3 methyl dC shows a
substantially increased metabolic stability which results in a
sustained antiviral activity. The compound exhibits a substantially
pronounced antiviral activity at non-cytotoxic concentrations.
5-trifluoromethyl-2'-deoxycytidine is prepared by reacting
5-trifluoromethyl-2'-deoxyuridine (F.sub.3 dU), in which the free
hydroxy groups have been protected, with ammonia. The reaction is
generally carried out at an elevated temperature which does not
exceed the decomposition temperatures of the starting materials or
the end products. The reaction temperature can generally vary from
about 50.degree. C. to 250.degree. C., and preferably from about
60.degree. C. to 100.degree. C. It has been found in practice that
temperatures of about 60.degree. C. to 80.degree. C. give most
satisfactory results. The precise temperature at which the reaction
is carried out will, of course, depend on the nature of the
reactants and the solvents employed, and the most appropriate
temperature can be readily determined by routine
experimentation.
It is generally necessary to protect the free hydroxy groups before
amination will proceed satisfactorily. It is possible to employ any
suitable protecting group, although for ease of handling it is
generally preferred to use a blocking or protecting group which
produces a crystalline rather than a liquid product. It has been
found that synthesis proceeds most satisfactorily using a silyl
protecting group such as the trimethylsilyl group, which can be
introduced according to the procedure described by Vorbruggen and
Niedballa, Angew. Chem. Internat. Edit. Volume 10, No. 9, 657
(1971), the disclosure of which is hereby incorporated by
reference. Thus, the reaction is preferably carried out by reacting
5-trifluoromethyl-2'-deoxyuridine (F.sub.3 dU) with a silylating
agent such as hexamethyldisilazane (HMDS) or trimethylsilylchloride
(trimethylchlorosilane or TMCS) in the presence of excess ammonia.
The silylating agent is usually employed in an excess and serves as
a solvent for the reaction as well as the silylating agent. It is
also possible to use different protecting groups on different free
hydroxy groups. For instance, the 2,4-positions of the pyrimidine
ring can be protected by reaction with one type of protecting group
agent, and the hydroxy groups on the deoxyfuranosyl ring can be
protected by use of a different protecting group.
The reaction is usually carried out for at least ten hours, more
usually twenty to fifty hours. It is not essential to conduct the
reaction under superatmospheric pressure, but it has been found
advantageous to conduct the reaction in a sealed tube or in an
autoclave to avoid undue loss of ammonia during the heating
process. When the reaction is carried out in an autoclave or sealed
tube, pressures of 50-200 psi, more preferably 60-80 psi, have
resulted in good yields of the desired compound F.sub.3 methyl
dC.
When the reaction is completed, the resulting reaction mixture is
usually an oily brown liquid which can be worked up according to
conventional techniques to yield the desired compound F.sub.3
methyl dC as a white crystalline solid. The compound F.sub.3 methyl
dC is insoluble in acetone and partially soluble in water, and can
be satisfactorily recrystallized from hot water.
A surprising and unexpected feature of the preparation is the
stability of the CF.sub.3 group under the reaction conditions. The
literature teaches that heating
5-trifluoromethyl-2'-deoxy-3',5'-di-O-toluyluridine with methanolic
ammonia in a steel bomb at about 100.degree. C. forms entirely the
5-carbomethoxynucleoside (Ryan et al., J. Org. Chem., 31, 1181
(1966). It is possible that the presence of a protecting group in
the pyrimidine ring changes the course of the reaction.
Another surprising and unexpected feature of the preparation of
F.sub.3 methyl dC is that the silylation/amination reaction does
not proceed in the absence of an N.sub.1 -substituent. Thus,
reaction of 5-trifluoromethyluracil with HMDS and excess ammonia
according to the reaction conditions described above does not yield
the corresponding amine compound, as illustrated by Comparative
Example A hereinbelow.
The starting compound, 5-trifluoromethyl-2'-deoxyuridine
(trifluorothymidine) can be prepared by procedures such as those
described in Heidelberger et al., J. Am. Chem. Soc., 34, 3597
(1962) and J. Med. Chem., 7, 1 (1964) and U.S. Pat. No. 3,201,387,
and Ryan et al., J. Org. Chem., 31, 1181 (1966).
In the silylation reaction described above, it has been found that
a mixture of HMDS and a small amount of TMCS will produce a higher
yield or faster reaction, as apparently a small amount of TMCS
produces a catalytic effect. This effect is disclosed in U.S. Pat.
No. 4,024,143, issued May 17, 1977, the disclosure of which is
hereby incorporated by reference for the teaching of silylation
reactions therein.
The preferred silylating agents have been described hereinabove.
Broadly speaking, the silylation reaction can be conducted using at
least a stoichiometric amount of a silylating agent which is:
silane of the formula
wherein R' is lower alkyl and X is halogen, and/or
disilazane of the formula
wherein R' is lower alkyl
at a temperature of about room temperature to the boiling point of
the reaction mixture. The lower alkyl groups of the above formulae
can contain from 1 to about 4 carbon atoms.
The authenticity of the compound of the present invention is
established by the following procedure wherein F.sub.3 dT is
trifluorothymidine (5-trifluoromethyl-2'-deoxyuridine).
F.sub.3 dT (R.sub.f =0.43) and F.sub.3 methyl dC (R.sub.f =0.80)
are separable in a chromatography system consisting of Whatman 3MM
and H.sub.2 O saturated n-butanol-NH.sub.3 (100 ml H.sub.2 O
saturated n-butanol+1 ml concentrated NH.sub.4 OH). Incubation of
F.sub.3 methyl dC with a crude source of cytidine deaminase extract
of human epidermoid carcinoma (HEP-2 cells) resulted in the
formation of an R.sub.f =0.43 spot and disappearance of the 0.80
spot on the above chromatography system. F.sub.3 dT incubated with
cytidine deaminase remained unchanged chromatographically.
The results of incubation with HNO.sub.2 (pH 4.5) at room
temperature were identical to those obtained above. Incubation of
F.sub.3 methyl dC for 9 hours resulted in approximately 98%
conversion to a product that has the same R.sub.f as F.sub.3 dT in
the same solvent system described above. F.sub.3 dT remained
unaltered. Incubation of F.sub.3 methyl dC and F.sub.3 dT with an
acetate buffer (pH 4.5) for 9 hours did not lead to modification of
the deoxyribonucleosides.
The following spots were eluted into H.sub.2 O and scanned from 225
to 350 nm: (a) standard, (b) F.sub.3 methyl dC standard, (d)
R.sub.f =0.43 spot after cytidine deaminase treatment of F.sub.3 dT
and (d) R.sub.f =0.43 spot after cytidine deaminase treatment of
F.sub.3 methyl dC. The u.v. absorption profile of F.sub.3 dT
derived from deamination of F.sub.3 methyl dC is identical to that
obtained from authentic F.sub.3 dT.
Solutions a, c, and d, above, were adjusted to the same O. D. and
used as substrates for HSV-2 induced pyrimidine deoxyribonucleoside
kinase. Results:
______________________________________ SOLUTION nmoles
phosphorylated/60min. ______________________________________ a 0.07
c 0.08 d 0.05 ______________________________________
Thus, the product of cytidine deaminase treatment of F.sub.3 methyl
dC (d) was phosphorylated to the same extent as F.sub.3 dT.
Stock solutions of F.sub.3 dT and F.sub.3 methyl dC were made up
and adjusted to the same concentration as the F.sub.3 dT and
F.sub.3 methyl dC purified chromatographically (solutions a and b,
respectively). Usually, F.sub.3 methyl dC is phosphorylated 1/6 of
the extent that F.sub.3 dT is phosphorylated. This experiment was
performed to determine if the chromatographic purification of
F.sub.3 methyl dC described above resulted in better
phosphorylation relative to F.sub.3 dT. This experiment was
performed twice. Results:
______________________________________ nmoles phosporylated/4 hrs
______________________________________ Standard F.sub.3 dT 0.48
(0.57 Solutions F.sub.3 methyl dC 0.48 (0.08) Approx 1/4 no
significant difference in ratios Chromatog. F.sub.3 dT 0.16 (0.19)
purified F.sub.3 methyl dC 0.04 (0.06) Approx 1/3 to 1/4
______________________________________
Values for chromatographically purified samples are probably lower
due to impurities arising from non-acid washed paper.
Other chromatographic systems and chemical analysis, including
thin-layer chromatography, can be utilized to confirm the
authenticity of the compound. The state of purity of the compound
tested in the determinations described above was approximately
80%.
The analysis for C.sub.10 H.sub.12 F.sub.3 N.sub.3 O.sub.4 was as
follows:
Calculated: C 40.67; H 4.06; N 14.23; Found: C 40.63; H 3.80; N
13.08.
F.sub.3 methyl dC exhibits surprisingly selective antiviral
activity, particularly against cells infected with HSV-1 and HSV-2
viruses, as well as against cells infected with
Herpes-varicella-zoster virus (VZV). Further, F.sub.3 methyl dC
exhibits a surprising and unexpected increase in metabolic
stability which results in low cell cytotoxicity, when used in
connection with a cytidine deaminase inhibitor, such as
tetrahydrouridine, as compared to other compounds, such as
trifluorothymidine, used with or without an inhibitor. The
combination of high antiviral activity and low cell cytotoxicity
results in the ability of F.sub.3 methyl dC to be used in such low
amounts, while still retaining effective antiviral activity, that
the cytotoxicity towards uninfected cells is minimized.
Because transformed cells express the Herpes encoded enzyme
activity and are selectively sensitive to F.sub.3 methyl dC, it is
expected that F.sub.3 methyl dC will possess the capacity to affect
latent infections which are severe problems that involve the
neurotropic aspects of these viruses.
F.sub.3 methyl dC may be formulated into pharmaceutical
compositions comprising, as the principal active ingredient,
pharmaceutically effective amounts of F.sub.3 methyl dC together
with a pharmaceutically acceptable carrier or diluent, for
intraperitoneal administration for animal studies, intravenous,
subcutaneous, intramuscular, oral or topical administration. The
concentration of the compound in the composition may vary from
about 0.01 to 50% by weight depending on the route of
administration, the frequency of administration, the severity of
the condition, the age, weight and general physical condition of
the patent being treated. When the composition is in the form
suitable for topical administration, for example a cream, the
concentration of F.sub.3 methyl dC will generally vary from about 5
to 50 wt.%, preferably about 5 to 20 wt.%, more preferably from
about 5 to 10 wt.%. When the composition is in the form suitable
for intraperitoneal administration for animal studies, for example,
an aqueous solution of F.sub.3 methyl dC, the concentration of
F.sub.3 methyl dC will generally vary from about 0.5 to 5% w/v,
more usually about 1% w/v. For oral administration, the
concentration of F.sub.3 methyl dC will generally be from 0.05 to
10 wt.%, preferably about 0.5 to 5 wt.%, and more preferably about
1 to 2 wt.%.
When F.sub.3 methyl dC is used for intravenous injection, the
concentration of the compound will vary from about 0.05 to about 5%
w/v, preferably about 0.1 to about 0.5% w/v. For intramuscular
injection, the same concentrations as described above for the
intraperitoneal mode of administration will be utilized.
Furthermore, in certain instances, such as for certain types of
encephalitis, intracranial injection may be utilized.
Other methods of administration may also be used. Suppositories may
be used for certain types of viral infections, and it is possible
that for some applications the F.sub.3 methyl dC will be
administered in the form of slow-release surgical implants.
The pharmaceutically acceptable carrier or diluent employed in the
compositions of the present invention may be any compatible
non-toxic material suited for mixing with the active compound
F.sub.3 methyl dC. When the composition is in a form suitable for
parenteral use, for example intramuscularly or intravenously, the
carrier which preferably is an aqueous vehicle, may also contain
other conventional additives, such as a suspending agent for
example methyl cellulose or polyvinylpyrrolidone (PVP), and a
conventional surfactant. For oral administration, the compositions
can be formulated as aqueous solutions, suspensions, capsules or
tablets, suitably containing appropriate carriers or diluents, for
example lactose, starch and/or magnesium stearate. In certain
instances, increased antiviral activity may be obtained by
coadministration of DMSO, which is also a solvent for the F.sub.3
methyl dC.
In order to inhibit the deaminating effect of enzymes such as
cytosine deaminase, with the consequent reduction in antiviral
activity, it is necessary for antiviral uses to coadminister either
previously or with the compound F.sub.3 methyl dC, a deamination
inhibiting agent, such as tetrahydrouridine (H.sub.4 U) or
2'-deoxytetrahydrouridine (H.sub.4 dU). Thus, the antiviral
pharmaceutical compositions comprise (as the principal active
ingredient) a pharmaceutically effective amount of F.sub.3 methyl
dC, together with inhibiting amounts of a cytidine deaminase
inhibitor, for example, tetrahydrouridine or
2'-deoxytetrahydrouridine. A pharmaceutically acceptable carrier or
diluent such as described above is generally present, depending on
the nature of the composition. Tetrahydrouridine and
2'-deoxytetrahydrouridine are not toxic in man at extremely high
concentrations. Furthermore, they are relatively metabolically
stable. The weight ratio of tetrahydrouridine or
2'-deoxytetrahydrouridine to F.sub.3 methyl dC can be 500:1 to 1:1,
but more usually will be about 20:1 to about 5:1.
To determine the toxicity of F.sub.3 methyl dC or other antiviral
agents to uninfected cells, non-confluent cultures of human
epidermoid laryngeal carcinoma (HEp-2) cells were treated with
nucleoside analogs at varying concentrations for 48 hours, at which
time the monolayer of cells is washed with phosphate buffered
saline in order to remove any residual analogs. The cells are then
trypsinized to remove them from the culture dishes and are replated
at various dilutions. At 7 days the cultures are stained and
colonies of 50 cells or greater are counted as one viable cell.
Viability, which is a valid parameter of toxicity is thus
determined by replating the cells. Therefore, toxicity is measured
in terms of cellular replication: colony formation.
FIG. 1 indicates the cytotoxicity of F.sub.3 methyl dC and F.sub.3
dT and without H.sub.4 U against HEp-2 cells (see Table I for
similar data for F.sub.3 methyl dC). The dramatic enhancement of
survival that is obtained when cells are grown in F.sub.3 methyl dC
and H.sub.4 U will be readily noted.
To demonstrate inhibition of viral replication, HSV types 1 or 2
are adsorbed to HEp-2 cells at low multiplicities for two hours at
37.degree. C. The culture medium containing the nucleoside analogs
at varying concentrations are added to the infected cultures. In
the case of HEp-2 cells which contain high deaminase levels,
H.sub.4 U is incorporated into the medium. At 48 hours, virus is
harvested from the cultures by freezing and thawing. The virus
produced from each culture is titred by plaque assay in BHK cells.
By using this protocol the antiviral effectiveness of F.sub.3
methyl dC can be compared directly with the effectiveness of other
antiviral agents. Furthermore, by including H.sub.4 U in the
medium, the effectiveness of F.sub.3 methyl dC can be determined
without deamination at the nucleoside level.
FIG. 2 demonstrates the antiviral activity of F.sub.3 methyl dC and
F.sub.3 dT, with and without H.sub.4 U against HSV-2. The results
indicate that the compounds are equally potent antiviral agents. An
examination of Table II or a comparison with FIG. 1, however,
reveals that F.sub.3 methyl dC (+H.sub.4 U) is only marginally
cytotoxic at concentrations that possess effective antiviral
activity, whereas F.sub.3 methyl dC (without H.sub.4 U) and F.sub.3
dT.+-.H.sub.4 U are extremely cytotoxic concentrations at which
they display antiviral activity. This then indicates a high
therapeutic index for F.sub.3 methyl dC and H.sub.4 U.
Table III indicates that when cells are grown on F.sub.3 methyl dC
about a 5-fold increased survival can be obtained with the use of
2'-deoxytetrahydrouridine (H.sub.4 dU) than with H.sub.4 U without
impairing the effectiveness of the antiviral activity of F.sub.3
methyl dC vs. HSV-2.
FIG. 3 indicates the antiviral activity of F.sub.3 methyl dC and
F.sub.3 dT with and without H.sub.4 U vs. HSV-1. Note that F.sub.3
methyl dC .+-.H.sub.4 U displays more potent antiviral activity
than F.sub.3 dT.+-.H.sub.4 U.
A summary of the cogent data is shown in Table IV. F.sub.3 methyl
dC.+-.H.sub.4 U is an effective antiviral agent at concentrations
that display marginal to only moderate cytotoxicity.
Animal patients, including humans, suffering from diseases caused
by Herpes and Herpes-like viruses can be treated by administering
to the patient a pharmaceutically effective amount of F.sub.3
methyl dC preferably in the presence of an deamination inhibitor
and optionally, but preferably in the presence of a
pharmaceutically acceptable carrier or diluent.
For the treatment of systemic infections, the F.sub.3 methyl dC of
the present invention will preferably be administered by
intravenous injection and less likely, but possibly, by oral
administration. For the case of topical infection, the F.sub.3
methyl dC will most likely be administered topically.
It has been found that particularly advantageous results are
obtained when the dosage of the compound F.sub.3 methyl dC to the
patient is from about 0.01 mmoles/kg to 0.25 mmoles/kg per day for
7 days. That is, 3 mg/kg body weight to 75 mg/kg body weight; for
example, about 10 mg/kg body weight per day for 7 days or 700 mg
for a 70 kg man per day for 7 days. These projections are based on
studies with the mouse in which it was found that a 60% survival
was obtained with 250 mg F.sub.3 methyl dC per kg once per day for
7 days with H.sub.4 U coadministered one hour prior to the F.sub.3
methyl dC. 100 percent survival was obtained with a dose of 50 mg.
F.sub.3 methyl dC per kg/day for 7 days when it was coadministered
with H.sub.4 U. The LD.sub.50 for F.sub.3 methyl dC (coadministered
with H.sub.4 U) was 325 mg/kg/day for 7 days.
Although the discussion above is centered on the compound F.sub.3
methyl dC, it will be appreciated that other analogs containing a
perfluorinated lower alkyl or lower alkenyl group in the
5-position, for example, 5-pentafluoroethyl-2'-deoxycytidine and
5-trifluorovinyl-2'-deoxycytidine, may exhibit similar antiviral
and chemotherapeutic activity. In addition, 2',3'-dideoxy F.sub.3
methyl cytidine may be a selective DNA chain terminator for Herpes
infected cells and the arabinosides 5-F.sub.3 thymine arabinoside
and 5-F.sub.3 methylcytosine arabinoside may also be used. In
addition, there may be potential efficacy in 2'-azido-5-F.sub.3
methyl dC for such uses.
EXAMPLES OF THE INVENTION
The present invention is further illustrated by the following
non-limiting Examples, wherein percentages are by weight unless
otherwise noted.
EXAMPLE I--Synthesis of F.sub.3 methyl dC
A mixture of 5-trifluoromethyl-2'-deoxyuridine (F.sub.3 dU) (10 g;
0.034 mole), hexamethyldisilazane (HMDS), (100 g) and ammonium
chloride (0.01 g) was saturated with ammonia and heated at
150.degree. C. overnight in a 500-ml Fischer-Porter aerosol
compatibility tube. A light brown clear solution was obtained and
the heating was continued for a further 24 hours. After a total of
44 hours, the heating was stopped and a clear brown solution was
obtained. The solvent was removed under partial vacuum using a
rotary evaporator at about 50.degree. to 60.degree. C. The residue
was refluxed with methanol (150 ml) for about 6 hours. The methanol
was removed using a rotary evaporator to give a solid (about 10 g).
The solid was dissolved in boiling water (250 ml), filtered and
cooled, giving a crystalline solid (2 g). Further crystals were
deposited from the mother liquor which were isolated and shown to
be the same as the crystals obtained earlier. The combined crystals
were refluxed in ethanol (30 ml) to give the desired compound
F.sub.3 methyl dC.
______________________________________ Elemental Analysis C H N
______________________________________ Actual 40.31 4.46 13.70
Theoretical 40.67 4.06 14.23
______________________________________
U.V. Data
______________________________________ .lambda. (0.1 HCl) = 282 m
.mu. (E = 10, 410) (max) .lambda. (0.1 NaOH) = 279 m .mu. (E = 10,
280) (max) ______________________________________
I.R. Data
3200 (broad), 1650 (broad), 1160, 1100, 1060 cm.sup.-1
EXAMPLE II
The experiment of Example I was repeated as follows using a mixture
of HMDS and trimethylsilyl chloride (TMCS) as a silylating agent. A
mixture of F.sub.3 dU (5 g), HMDS (50 ml) and TMCS (0.2 ml) in a
100 ml autoclave was saturated with ammonia (10 g) at room
temperature. A slight pressure of 10-20 psi was recorded. The
mixture was then heated at about 165.degree. C. for about 48 hours
with a recorded pressure of about 200 psi. The autoclave was then
opened and the resulting mixture was poured into a 50-ml flask. The
solvent was removed under vacuum leaving a brown viscous liquid.
Thin layer chromatography using a water solution indicated that
only a trace of the starting materials remained and that the major
component of the mixture was a different compound. The mixture was
extracted with boiling water (200 ml), decolorized with charcoal,
and filtered. The water was removed under vacuum using a rotary
evaporator to give about 0.15 to 0.2 g of a compound which was
shown by analysis to be the same compound as obtained in Example
I.
EXAMPLE III
The procedure of Example II was repeated except that the reaction
was performed in a sealed tube instead of an autoclave. A mixture
of F.sub.3 dU (3.0 g) and HMDS (2.8 g) was placed in a
Fischer-Porter tube and saturated with ammonia for about 30
minutes. A gel was formed. Then trimethylsilyl chloride (0.2 ml)
was added to the mixture, and the tube was sealed and heated to
140.degree. C. overnight, giving a pressure of 60 psi. The
temperature was then increased to 150.degree. C. giving a pressure
of 80 psi. The heating was continued for a further 24 hours. A
clear light brown solution was obtained and some solid had sublimed
onto the cooler parts of the tube. The tube was cooled and some
solid precipitated. The tube was opened and cooled and the HMDS was
removed under vacuum at 50.degree. to 60.degree. C. leaving a
viscous brown residue. Methanol (50 ml) was added and the mixture
was heated to 65.degree. to 70.degree. C. for 6 hours. The methanol
was then removed under vacuum leaving a brown solid which was
dissolved in hot water (200 ml) and filtered. The filtrate was
decolorized with charcoal, filtered and evaporated to dryness,
giving a solid (2.5 g). This solid was dissolved in boiling water,
filtered and cooled, yielding crystals (0.1 g) which were shown by
analysis to be the same compound as obtained in Examples I and
II.
EXAMPLE IV
An aqueous solution of the compound F.sub.3 methyl dC was prepared
by dissolving F.sub.3 methyl dC (about 0.2 gm) in physiologically
pure water (5 ml) under sterile conditions. The solution which was
suitable for administration by injection, was then sealed in
ampoules and stored, ready for future use.
EXAMPLE V
A formulation of the compound F.sub.3 methyl dC suitable for
topical administration was prepared by compounding F.sub.3 methyl
dC (0.4 gm) with lanolin (1 gm) as a carrier in a conventional
manner to form a cream of smooth consistency, suitable for topical
administration.
EXAMPLE VI
A mixture of 5-trifluoromethyl-2'-deoxyuridine (F.sub.3 dU) (18 g),
HMDS (200 ml) and TMCS (1.5 ml) in a Fischer-Porter bottle was
pressurized with anhydrous ammonia to 40 psi. The stirred mixture
was heated at 65.degree. to 75.degree. C. for 94 hours, giving a
pressure of 60 to 70 psi. The excess ammonia was vented off and the
excess HMDS was removed under vacuum. Methanol was added to the
residue and the mixture was heated to reflux temperature. The
methanol was removed under vacuum and the solid residue was
recrystallized from water yielding F.sub.3 methyl dC (7 g)
identical to that obtained in Example I.
______________________________________ Analysis C H N
______________________________________ Actual 40.72 4.23 14.36
Theoretical 40.67 4.06 14.23
______________________________________
COMPARATIVE EXAMPLE A
A mixture of 5-trifluoromethyluracil (0.3), HMDS (5 ml) and TMCS
(0.2 ml) in a Fischer-Porter tube was pressurized with anhydrous
ammonia to 18 psi. The mixture was heated at
160.degree.-170.degree. C. for 72 hours, giving a pressure of 60
psi. After removal of excess HMDS and hydrolysis of the reaction
product with excess methanol under reflux, the starting material
(5-trifluoromethyluracil) was recovered. There was no evident (TLC)
of other products.
TABLE I
__________________________________________________________________________
CYTOTOXICITY OF F.sub.3 METHYL dC vs HEp-2 CELLS % Survival
-H.sub.4 U +H.sub.4 U Concentration mM .003 .03 .3 .003 .03 .3
__________________________________________________________________________
Experiment 1 3 <.03 <.03 100 57 <.03 2 0.1 0.0005
<0.0005 93 15 <0.0005 3 0.1 0.0008 0.0007 53 25 0.001 4 0.6
0.02 -- 61 31 -- Average %S 0.95 <0.01 <0.01 77 32 <.01
__________________________________________________________________________
H.sub.4 U concentration: 100 .mu.g/ml in experiment 1 and 2 500
.mu.g/ml in experiment 3 and 4 H.sub.4 U does not display any
cytotoxic activity ##STR3##
TABLE II
__________________________________________________________________________
ANTIVIRAL ACTIVITY OF F.sub.3 METHYL dC vs HSV-2 Log.sub.10
Surviving Fraction -H.sub.4 U +H.sub.4 U Concentration mM .003 .03
.3 .003 .03 .3
__________________________________________________________________________
Experiment 1 -0.1 -3.1 -0 -3.0 2 -0.92 -2.7 -3.7 -1.5 -3.7 -3.7 3
-1.8 <-4.1 -1.9 <-4.1 4 -2.9 -3.7 Average Log.sub.10 S.F.
-0.94 -3.2 -3.7 -1.7 -3.6 -3.7 %S (from Table I) 0.95 <0.01
<0.01 77 32 <0.01
__________________________________________________________________________
H.sub.4 U concentration: 100 .mu.g/ml in experiment 1 and 2 500
.mu.g/ml in experiment 3 and 4 H.sub.4 U does not display any
antiviral activity Log.sub.10 S.F. = Log.sub.10 Surviving Fraction
of Plaque Forming Units ##STR4##
TABLE III ______________________________________ CYTOXICITY AND
ANTIVIRAL ACTIVITY OF F.sub.3 METHYL dC WITH AND WITHOUT H.sub.4 U
and 2'dH.sub.4 U No Addition +H.sub.4 U +2'dH.sub.4 U
______________________________________ % S of HEp-2 cells:
<0.006 9 .+-. 3 50 .+-. 5 0.06 mM Log.sub.10 S.F. HSV-2: -3.3
-3.4 -3.5 0.03 mM H.sub.4 U and 2'dH.sub.4 U: 500 .mu.g/ml
______________________________________
TABLE IV ______________________________________ RELATIONSHIP
BETWEEN THE CYTOTOXICITY OF F.sub.3 METHYL dC + H.sub.4 U AND ITS
ANTIVIRAL ACTIVITY vs HSV-1 AND HSV-2 IN CELL CULTURE CONCENTRATION
F.sub.3 methyl dC mM .003 .03 .3
______________________________________ % S 77 32 <.01 (from
Table I) Log.sub.10 S.F. vs HSV-2 -1.7 -3.6 -3.7 (from Table II)
Log.sub.10 S.F. vs HSV-1 -4.2 <-4.6 -- (from FIG. 3)
______________________________________
* * * * *